Background CD40-CD40Ligand interaction is a crucial costimulatory factor in T-cell mediated B-cell activation. Intervention into this system allows efficiant modulation of immune respone in transplantation and autoimmunity e.g. systemic lupus erythematosus (SLE). Recently CD40L (CD154) has been described as a novel antigen on activated platelets with impact towards thrombus formation and interplay with endothelial cells. Altered platelet CD40 L expression was already described in procoagulatory situations like instable angina pectoris.
Objectives As platelet activation by phospholipid antibodies is found in SLE and especially in Antiphospholipid syndrome (APS) we analysed CD40L expression in SLE and secondary APS.
Methods Using flowcytometric whole blood and platelet rich plasma double staining assay systems we analysed CD40 L expression. PE labelled CD40L (cloneTRAP1, Pharmingen) was employed, FITC-CD42b as detection system for platelets. 40 patients with diagnosis of SLE were included into this study, among them 10 with secondary APS and compared to 15 normal healthy donors (NHD). Results were analysed using Cellquest software (Becton Dickinson, San Jose, USA) differrenciating single from aggregated platelets under nonstimulatory and stimularory conditions (thrombin 30 uM, ADP 10 uM).
Results We found that CD 40 L expression was higher on aggregated compared to single platelets, where fluorescence means were close to baseline. We compared the whole blood method with platelet rich plasma and found lower background activity in whole blood test. Using established stimulators of platelets we could increase CD40L expression on platelets, espescially on aggregated platelets. Analysing CD40L expression in SLE patients we found no significant difference between SLE and NHD, neither on single or on aggregated platelets nor upon stimulation with ADP or thrombin. High CD40 L expression was observed in APS with highly significant differences compared to NHD and SLE analysing single platelets ((NHD < APS (p < 0,0001) SLE < APS (p < 0,0001)) as well as aggregated platelets. Upon stimulation with thrombin or ADP results were comparable indicating significantly higher CD40 L expression on platelets.
Conclusion We first describe high CD40 L expression on single and aggregated platelets in APS establishing CD40L as a novel platelet activation marker in APS. Interplay between CD40L on platelets and endothelial cells may contribute to procoagulatory conditions in APS. On the other hand we could not show altered CD40L expression in patients with SLE without clinical APS.
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